The discovery that Ras proteins are farnesylated and that inhibition of farnesylation prevents cellular transformation has generated an explosion of interest in this post-translational modification;currently, several drug candidates that target farnesyl transferase are in Phase 3 trials. However, despite this success, our current understanding of the enzymology of prenyltransferases remains incomplete. Moreover, many aspects concerning the biological function of protein prenylation remain unclear. In the last three years of this project, we have gained significant insights into the structure and mechanism of prenyltransferases;we have also made progress in understanding functional aspects of this modification. The specific aims of this renewal application are to: (1) Construct transition state structural models from kinetic isotope effect measurements for the reactions catalyzed by protein farnesyl and geranylgeranyl transferase;(2) Probe for intermediates in the reaction catalyzed by protein farnesyltransferase using caged forms of farnesyldiphosphate in conjunction with time-resolved crystallography;(3) Identify active site residues in the protease, RCE1;(4) Develop proteomic methods for determining the prenylation state of proteins and use them to measure prenylation states and levels in cancer cells treated with inhibitors of protein prenylation;(5) Augment the affinity of peptides that bind to RhoGDI and study the mechanism by which they cause cell death;(6) Study the effect of prenyl group structure on subcellular localization of prenylated peptides and proteins. Completion of the above aims should provide additional insights into the mechanism and function of protein prenylation